Project Summary/Abstract: Herpes simplex virus 1 (HSV-1) infects an estimated 3.7 billion people under the age of 50 worldwide. The primary symptoms of infection are orolabial lesions, but clinically severe diseases such as ocular keratitis and encephalitis can result, particularly in immunocompromised individuals. The ability to alternate between lytic replication in the epithelia and a largely dormant latency in neurons underpins HSV-1?s wide-spread prevalence. To replicate and spread, HSV-1 must both counteract and co-opt the host cell epigenetic machinery. Although viral DNA within the virion is not associated with histones, HSV-1 DNA is rapidly assembled into nucleosomes upon entry into the host nucleus. For productive lytic replication to occur, viral proteins must enable the transition of viral chromatin from this initial repressive chromatin state to a more permissive, de-condensed state. This proposal will investigate a novel mechanism by which HSV-1 navigates this reorganization of viral chromatin. Although chromatin-modifying and histone-modifying enzymes with pro-viral functions have been identified, there have been few histone variants linked to enhanced virus production. We recently identified the variant histone H2A.Bbd as a novel HSV-1 host factor, with H2A.Bbd depletion reducing wild-type HSV-1 yields. Incorporation of this histone variant into cellular nucleosomes is associated with both actively transcribed genes and a permissive chromatin state. H2A.Bbd is also targeted to and enriched at sites of host DNA synthesis. Our initial studies suggest that H2A.Bbd acts in HSV-1 lytic replication to enhance viral DNA synthesis. Furthermore, the H2A.Bbd histone chaperone NAP1L1 was identified previously as associating with the HSV-1 DNA replication protein ICP8. The established association of H2A.Bbd incorporation with de-condensed chromatin suggests that its incorporation into HSV-1 chromatin may increase viral DNA accessibility, potentially enhancing the access of HSV-1 DNA to viral replication proteins. We hypothesize that the histone variant H2A.Bbd promotes viral DNA synthesis during lytic replication by NAP1L1-mediated incorporation into HSV-1-associated nucleosomes to establish a permissive chromatin state. This proposal seeks to address whether H2A.Bbd is recruited to replication foci to promote viral DNA synthesis (Aim 1) and whether H2A.Bbd is incorporated into HSV-1-associated nucleosomes by NAP1L1 to alter viral chromatin and promote viral DNA accessibility (Aim 2). Aim 1 will test if total viral DNA synthesis levels are reduced in H2A.Bbd-depleted cells by qPCR (Aim 1.1); investigate recruitment of H2A.Bbd to replication foci by microscopy (Aim 1.2); and compare the association of replication proteins with viral DNA in the presence and absence of H2A.Bbd by iPOND-MS (Aim 1.3). Aim 2 will determine if H2A.Bbd is incorporated into viral chromatin by ChIP (Aim 2.1) and examine the impact of H2A.Bbd on viral DNA accessibility by ATAC-Seq (Aim 2.2). The proposed project aims to provide new insights into the epigenetic mechanisms underlying viral DNA synthesis, while potentially revealing new strategies for inhibiting HSV-1 and related herpes viruses via the therapeutic manipulation of viral chromatin.